Although several compounds with cyanide antidote activity exist, all function via stoichiometric reaction with free cyanide to form a less-toxic cyanide complex. As such, these cyanide scavengers only work at stoichiometric doses and only prior to cyanide interacting with its target proteins. A cyanide countermeasure that did not rely solely on scavenging of free cyanide would be a highly-valuable addition to the countermeasure arsenal because it could potentially 1) be effective at sub-stoichiometric doses, 2) be used in combination with existing scavenging agents, and 3) provide benefit even after cyanide had bound to its cellular targets. Recent discovery efforts within our consortium have identified glyoxylate as a potent and highly effective cyanide countermeasure. Treatment with glyoxylate rescues zebrafish, mice, and rabbits exposed to cyanide. Remarkably, glyoxylate does not appear to be functioning only as a cyanide scavenger. Rather, it appears to produce a rapid and dramatic metabolic transformation that normalizes several key metabolic derangements induced by cyanide, including perturbations in oxygen consumption, metabolite flux, and redox balance. Thus, glyoxylate appears to function through a mechanism that is very different from existing scavenger-based cyanide countermeasures. As such, it may complement existing countermeasures and provide a completely novel means of reversing cyanide's effects. In this project we will focus on delivering one or more glyoxylate-based countermeasure products. Efforts will include optimization of glyoxylate derivatives for use as cyanide antidotes through standard formulation and optimization strategies. We will also explore LDH, the presumptive glyoxylate target, as a potential source of second-generation cyanide antidotes. Finally, we will test the ability of glyoxylate to rescue the effects of other metabolic poisons, sulfide and azide. Specific aims include: Aim 1. To optimize glyoxylate as a countermeasure for clinical deployment. This aim focuses on formulation of glyoxylate to produce a stable countermeasure product that can be delivered by autoinjector. Aim 2. To develop alternative LDH substrates as second-generation, glyoxylate-like countermeasures. This aim explores a series of alternative LDH substrates as potential cyanide countermeasures through studies in zebrafish, mice, and rabbits. Aim 3. To test glyoxylate as a countermeasure for other metabolic poisons. This aim tests the ability of glyoxylate to reverse the toxic effects of other metabolic poisons including sulfide and azide. Successful completion of this project will deliver at least one fully validated, glyoxylate-based cyanide countermeasure that meets the BARDA requirements for advanced development. It will also fill the pipeline with second-generation countermeasures that exploit metabolic modulation to counteract cyanide toxicity. .